Abstract Gram-negative pathogens use outer membrane vesicles (OMVs) to kill host cells and competitors, avoid and interfere with the immune response, transfer antibiotic resistance genes, sequester and destroy antibiotics, and traffic both cell-to-cell communication signals and small RNAs. Despite their direct contribution to so many pathogenesis-related behaviors, our understanding of how OMVs are produced remains surprisingly incomplete. Several models of OMV biogenesis have been proposed using different organisms and identifying inputs from varying cellular pathways. Our team studies OMV biogenesis at the molecular level and uses this approach to discover features that drive OMV formation regardless of the input signal. Our Bilayer-Couple model describes how intercalation of a self-produced small molecule preferentially into the outer leaflet of the membrane causes it to expand relative to the inner leaflet and thereby induce membrane curvature. This framework can already explain models that have subsequently been put forth in other species. This proposal aims to unify disparate and species-specific models by expanding our understanding of molecular interactions between small molecule OMV inducers and outer membrane lipids. We will integrate molecular simulation and physical experiment to discover the role of chemical variations in controlling molecule-lipid association and curvature induction. We will exploit our recently developed all-atom molecular dynamics model of the outer membrane to make atomistic-level predictions about how different molecule derivatives drive curvature induction in membranes with different lipid chemotypes. Our established experimental approaches for assessing OMV production from different bacterial species and mutants will be used to test the in silico predictions. In the context of our recent discovery that many Gram-negative organisms, including important clinical pathogens, secrete factors to induce cross-species stimulation of OMV production, we will pursue two specific aims to characterize how variations in the chemical structure of an OMV inducer molecule influences its specific interactions with different outer membrane lipids that are found across species: (1) Characterize nanoscale interactions of OMV-inducing factors and molecular derivatives with outer membrane lipids and quantify membrane response to the interactions in all-atom simulations, (2) Make use of specific mutants and related species to test the predictions of the in silico model and demonstrate control of OMV biogenesis by small molecule-lipid interactions in vitro. The findings of the proposed work will help us bring together parallel and species-specific ideas about OMV biogenesis into a fundamental and robust model that can generalize across species. We will gain an important understanding about how pathogens interact to manifest negative clinical consequences and move toward the larger goal of tailoring treatments to disrupt detrimental pathogen-pathogen interactions that are mediated by OMVs.